U.S. patent number 5,631,132 [Application Number 08/429,523] was granted by the patent office on 1997-05-20 for nucleic acid probes and methods for detecting candida glabrata dna in blood.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Brent Lasker, Timothy J. Lott, Christine J. Morrison, Errol Reiss, Sandra Zakroff.
United States Patent |
5,631,132 |
Lott , et al. |
* May 20, 1997 |
**Please see images for:
( Certificate of Correction ) ** |
Nucleic acid probes and methods for detecting Candida glabrata DNA
in blood
Abstract
Provided is an isolated double-stranded nucleic acid consisting
essentially of the nucleotide sequences defined in the Sequence
Listing by SEQ ID NOs: 5-9. These are the ITS2 sequences for C.
albicans, C. parapsilosis, C. tropicalis, C. glabrata and C.
krusei. A method of diagnosing systemic candidiasis in a subject is
also provided. The method comprises the steps of: (a) collecting
blood from the subject into tubes containing detergent,
polypropylene glycol, sodium poyantholesulfonate, and sodium
ethylene diamine tetraacetic acid; (b) lysing Candida cells using
ZYMOLYASE.RTM.-100T with agitation; (c) extracting and
precipitating the DNA from the lysed cells; (d) amplifying the
precipitated DNA using universal fungal primer pairs derived from
the internal transcribed spacer regions of the Candida ribosomal
DNA; and (e) detecting amplified DNA from Candida by hybridizing
the amplified DNA with a probe that selectively hybridizes with
Candida DNA, the presence of amplified DNA indicating systemic
candidiasis.
Inventors: |
Lott; Timothy J. (Atlanta,
GA), Morrison; Christine J. (Atlanta, GA), Reiss;
Errol (Chamblee, GA), Lasker; Brent (Atlanta, GA),
Zakroff; Sandra (Clarkston, GA) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
|
[*] Notice: |
The portion of the term of this patent
subsequent to June 20, 2012 has been disclaimed. |
Family
ID: |
22065521 |
Appl.
No.: |
08/429,523 |
Filed: |
April 26, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
65845 |
May 20, 1993 |
5426027 |
|
|
|
Current U.S.
Class: |
435/6.12;
536/23.1; 536/24.3 |
Current CPC
Class: |
C12Q
1/6806 (20130101); C12Q 1/6895 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 001/68 (); C07H 021/02 ();
C07H 021/04 () |
Field of
Search: |
;436/6
;536/22.1,23.1,24.3 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Zakroff et al. Abstract No. 100930 Amer. Soc. for Microbiol. Gen.
Meeting, Atlanta, GA May 1993. .
Lott et al. Abstract Amer. Soc. for Microbiol. Gen. Meeting,
Atlanta, GA May 1993. .
Niesters et al. J. Clin. Microbiol. 31(4):904-910, Apr. 1993. .
Hopfer et al. J. of Med. and Vet. 31:65-75, 1993. .
Kan/Bennett Abstract 1627, Interscience Conf. on Anti-Microbial
Agents Chemotherapy, Oct. 1992. .
Rand/Houck Abstract 1628, Interscience Conf. on Anti-Microbial
agents Chemotherapy, Oct. 1992. .
Crampin, A.C. Abstract, J. Med. Microbiol. 37 (supp. 1) Abst. No.
283, Jul. 1992. .
Lehmann et al. Abstract F-16, Gen. Meet. Am. Soc. Microiol. New
Orleand, LA, May 1992. .
Miyakawa et al. J. of Clin. Microbiol. 30(4): 894-900, Apr. 1992.
.
Oren et al. Abstract D-46 Gen. Meeting Am. soc. for Microbiol.,
Dallas, TX, May 1991. .
Lott/Kuykendall Abstract F-78 Gen. Meeting Am. soc. for Microbiol.,
Dallas, TX May 1991. .
Barns et al. J. of Bacteriol. 173(7-8): 2147-2731, 1991. .
Jones, J.J. Clin. Microbiol. Rev. 3:32-45, 1990. .
Buchman et al. Surgery 108: 338-347, 1990. .
White et al. PCR Protocols: A Guide to Methods and Applications
Innis et al. Eds. Academic Press, Inc. pp. 315-322, 1990. .
Hendricks et al. System. Appl. Microbiol. 12: 223-229, 1989. .
Yue et al. Abstract, Clinical Research 37(2): 446A, 1989. .
Huffman et al. Authentication of ATCC Strains in the
Saccharomyces-Cerevisiae Complex by PCR Fingerprinting, Exp Mycol
16(4): 316-319, 1992..
|
Primary Examiner: Zitomer; Stephanie W.
Assistant Examiner: Whisenant; Ethan
Attorney, Agent or Firm: Needle & Rosenberg, P.C.
Parent Case Text
This application is a division of application Ser. No. 08/065,845,
filed May 20, 1993 which status is now U.S. Pat. No. 5,426,027.
Claims
What is claimed is:
1. An isolated double stranded nucleic acid consisting of the
nucleotide sequence defined in the Sequence Listing by SEQ ID NO:
8.
2. An isolated nucleic acid of up to 231 nucleotides that
specifically hybridizes with the nucleic acid of claim 1.
3. A method of diagnosing systemic candidiasis in a subject
comprising the steps of:
a) collecting blood from the subject into tubes containing
detergent, polypropylene glycol, sodium polyanetholesulfonate, and
sodium ethylene diamine tetraacetic acid;
b) lysing Candida cells using ZYMOLASE.RTM.-100T with
agitation;
c) extracting and precipitating the DNA from the lysed cells;
d) amplifying the precipitated DNA using universal fungal primer
pairs that amplify the internal transcribed spacer regions of the
Candida ribosomal DNA; and
e) detecting amplified DNA from as few as 1 Candida glabrata cell
per 100 microliters of blood by hybridizing the amplified DNA with
a probe that specifically hybridizes with the nucleic acid of claim
1, the presence of hybridization indicating systemic
candidiasis.
4. The method of claim 3, wherein the lysis step further uses the
lysis buffer from the ISOQUICK.RTM. kit.
5. The method of claim 3, wherein the agitation is by rocking at 16
cycles per minute.
6. The method of claim 3, wherein the extracting step uses the
extraction matrix in the ISOQUICK.RTM. kit.
7. The method of claim 3, wherein one of the primers of the primer
pair is derived from the internal transcribed spacer 1 and the
other primer of the primer pair is derived from the internal
transcribed spacer 2.
8. The method of claim 3, wherein one of the primers of the primer
pair is derived from the internal transcribed spacer 3 and the
other primer of the primer pair is derived from the internal
transcribed spacer 4.
9. The method of claim 3, wherein the detecting step hybridization
is by dot blot hybridization.
Description
BACKGROUND OF THE INVENTION
Candida albicans is a commensal of the gastrointestinal tract. C.
albicans, and to a lesser extent several other related species, are
of increasing importance as opportunistic pathogens in
immunocompromised hosts. A dimorphic, diploid yeast with no known
sexual cycle, C. albicans is an endogenous organism that can be
isolated from skin and mucosal tissues of persons whose immune
systems are intact. However, perturbations of the immune or
endocrine systems can create opportunities for Candida species to
convert from a commensal state to invade tissues either locally or
systemically. An example of this opportunism is the oral-esophageal
or vaginal candidiasis that is encountered in association with HIV
infection. In C. albicans, the nuclear rDNA genes encoding the 5S,
18S, 5.8S, and 28S rRNAs are found as 50-100 copy tandem repeats of
approximately 10 kb unit length on chromosome seven (Magee et al.,
1987, Thrash-Bingham and Gorman, 1992). The 5S rDNA gene (121 bp)
is flanked by two nontranscribed regions located between the small
and large subunits, and collectively termed the intergenic spacer
(IGS). Ribosomal 5.8S sequences have been compiled from a variety
of eukaryotes (Dams et al., 1988). In addition, sequence analysis
of the 5.8/28S internally transcribed spacer (ITS) region has shown
strain variation within at least one fungal species (O'Donnell,
1992), while other species have demonstrated complete conservation
(Mitchell et al., 1992). Strain-specific restriction polymorphisms
(RFLPs) have previously been observed in the IGS region for C.
albicans (Magee et al., 1987).
An opportunistic fungus, C. albicans also causes systemic disease
in severely immunocompromised hosts. It is the most causative
species of disseminated candidiasis followed by C. tropicalis, C.
parapsilosis, and C. glabrata (Odds, 1988). Dissemination occurs
when Candida is spread via the bloodstream or by invasion of
mucosal surfaces to internal organs (Odds, 1988). High-risk patient
populations include individuals with malignancy or neutropenia,
those receiving chemotherapy and/or multiple antibiotics, and those
with indwelling catheters or low birth weight infants (Armstrong,
1989).
Diagnosis of systemic candidiasis is complicated by the absence of
clinically distinguishing signs, frequently negative blood
cultures, and the absence of a reliable serological test to detect
infection. Currently, disseminated candidiasis is often diagnosed
by a minimum of at least two positive blood cultures (Odds, 1988).
However, blood culture alone is clearly not sufficient for the
diagnosis of disseminated candidiasis since as many as 50% of
disseminated candidiasis cases are diagnosed at autopsy (Telenti,
et al. 1989). The nephrotoxicity of amphotericin B, the drug of
choice for immunocompromised patients with disseminated disease,
precludes its use for prophylaxis.
These facts, in conjunction with the difficulty of reliably
culturing Candida from the blood and the lack of a sensitive and
specific serological test to detect disease, underscore the need to
develop alternative diagnostic approaches.
Technology has been developed for the detection of bacterial and
viral DNA from the bloodstream of infected patients through the use
of the polymerase chain reaction (PCR). The PCR amplifies genomic
DNA geometrically so that it may be detected by agarose gel
electrophoresis, Southern blotting, or dot blot hybridization
(Miyakawa et al. 1992, Kafatos et al. 1979, Lasker et al.
1992).
PCR-based diagnostic methods may provide increased sensitivity
relative to blood culture techniques since viable organisms are not
required for amplification or detection. There has only been one
report to date describing the detection of C. albicans cells in
infected patient blood through the use of PCR-amplified DNA
(Buckman et al. 1990). Buchman et al. lysed C. albicans cells with
ZYMOLYASE and proteinase K and extracted the DNA with phenol and
chloroform. The limit of sensitivity by this method was 120 cells
per ml of whole blood. As described, this method was time
consuming, labor-intensive, repeatedly used toxic chemicals (phenol
and chloroform), and has not been shown to be readily reproducible.
In addition, a single copy gene, the cytochrome P-450 gene, was the
target for DNA amplification, thus making the method much less
sensitive. Miyakawa et al. described improved sensitivity by use of
Southern blot hybridization for the detection of PCR products from
Candida DNA (Miyakawa et al. 1991). The limit of sensitivity by
Southern blot in their study was 10 cells per ml of urine and did
not address detection in blood.
The ability to detect Candida in blood is crucial for the rapid and
accurate diagnosis of systemic candidiasis, because detection from
urine or mucosal secretions can be confused with the normal
commensal status of the organism or a localized non-disseminated
infections. The present invention provides a rapid method for the
isolation, release, purification and amplification of C. albicans
DNA from blood and other body fluids of infected patients. This
method minimizes the use of phenol and chloroform and uses
universal fungal primers to the multi-copy ITS region of rDNA, to
enhance detection of Candida DNA. The invention provides a rapid
approach to species identification through the use of non-conserved
regions of the ITS2 flanked by highly conserved, functional
domains.
SUMMARY OF THE INVENTION
The present invention provides an isolated double-stranded nucleic
acid consisting essentially of the nucleotide sequence defined in
the Sequence Listing by SEQ ID NO:5. This is the C. albicans ITS2
sequence and includes a nucleic acid comprising a nucleotide
sequence that is specific for C. albicans. Further examples of an
isolated double stranded nucleic acid of the present invention
consist essentially of the nucleotide sequences defined in the
Sequence Listing by SEQ ID Nos: 6-9. These are the ITS2 sequences
for C. parapsilosis, C. tropicalis, C. glabrata and C. krusei.
These nucleic acids can include a nucleotide sequence that is
specific for the respective organism.
An isolated nucleic acid that specifically hybridizes with or
selectively amplifies a nucleic acid of the invention or fragments
thereof is also contemplated. An isolated nucleic acid
complementary to the above nucleic acid is also provided.
A method of diagnosing systemic candidiasis in a subject is also
provided. The method comprises the steps of: (a) collecting blood
from the subject into tubes containing detergent, polypropylene
glycol, sodium polyanetholesulfonate, and sodium ethylene diamine
tetraacetic acid; (b) lysing Candida cells using
ZYMOLYASE.RTM.-100T with agitation; (c) extracting and
precipitating the DNA from the lysed cells; (d) amplifying the
precipitated DNA using universal fungal primer pairs derived from
the internal transcribed spacer regions of the Candida ribosomal
DNA; and (e) detecting amplified DNA from Candida by hybridizing
the amplified DNA with a probe that selectively hybridizes with
Candida DNA, the presence of amplified DNA indicating systemic
candidiasis.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an isolated double-stranded nucleic
acid consisting essentially of the nucleotide sequence defined in
the Sequence Listing by SEQ ID NO: 5. This includes the C. albicans
ITS2 sequence. By "isolated" is meant separated from other nucleic
acids found in the naturally occurring organism. The nucleic acid
comprises a nucleotide sequence that is specific for C. albicans.
By "specific" is meant a sequence which does not hybridize with
other nucleic acids to prevent determination of an adequate
positive hybridization with nucleic acids from C. albicans. Probes
which "specifically hybridize" with the double-stranded nucleic
acid are hybridizing with one of the two strands when in single
stranded form.
A further example of an isolated double stranded nucleic acid of
the present invention consists essentially of the nucleotide
sequence defined in the Sequence Listing by SEQ ID NO: 6. This
includes the ITS2 sequence for C. parapsilosis. This nucleic acid
comprises a nucleotide sequence that is specific for C.
parasilosis.
Another example of the isolated double stranded nucleic acid of the
invention consists essentially of the nucleotide sequence defined
in the Sequence Listing by SEQ ID NO: 7. This includes the C.
tropicalis ITS2 sequence. This nucleic acid comprises a nucleotide
sequence that is specific for C. tropicalis.
A still further example of the isolated double stranded nucleic
acid of the invention consists essentially of the nucleotide
sequence defined in the Sequence Listing by SEQ ID NO: 8. This
includes the C. glabrata ITS2 sequence. This nucleic acid comprises
a nucleotide sequence that is specific for C. glabrata.
Another example of the isolated double stranded nucleic acid of the
invention consists essentially of the nucleotide sequence defined
in the Sequence Listing by SEQ ID NO: 9. This includes the C.
krusei ITS2 sequence. This nucleic acid comprises a nucleotide
sequence that is specific for C. krusei.
An isolated nucleic acid that specifically hybridizes with or
selectively amplifies a nucleic acid of the invention or fragments
thereof is also contemplated. An isolated nucleic acid
complementary to the above nucleic acid is also provided. The
sequences can be selected based on the nucleotide sequence and the
utility of the particular sequence. More specifically the invention
provides isolated nucleic acids that specifically hybridize with
the nucleic acids consisting essentially of the nucleotide
sequences defined in the Sequence Listing by SEQ ID NOs: 5-9.
The term "consisting essentially of", as used herein includes
modifications to the nucleic acids of the invention as long as the
specificity (genus or species) of the nucleic acids is maintained.
Likewise, fragments used as primers or probes can have
substitutions so long as enough complementary bases exist for
specific hybridization (Kunkel et al. Methods Enzymol. 1987:
154:367, 1987).
The nucleic acid can have homology with nucleotide sequences
present in more than one Candida species. Such a nucleic acid
sequence shared with other Candida species can be used, for
example, as a primer to simultaneously amplify nucleic acids from
more than one Candida species. The amplified nucleic acids can then
be detected using the specific nucleic acids described herein to
permit either genus specific or species specific diagnosis. Thus,
the specific nucleic acid can be specific for the genus Candida and
can be used to detect any candidiasis in methods such as polymerase
chain reaction, ligase chain reaction and hybridization.
A method of diagnosing systemic candidiasis in a subject is also
provided. The method comprises the steps of: (a) collecting blood
from the subject into tubes containing detergent, polypropylene
glycol, sodium polyanetholesulfonate, and disodium ethylene diamine
tetraacetic acid ((Na).sub.2 EDTA); (b) lysing Candida cells using
zymolase-100T with agitation; (c) extracting and precipitating the
DNA from the lysed cells; (d) amplifying the precipitated DNA using
universal fungal primer pairs derived from the internal transcribed
spacer regions of the Candida ribosomal DNA; and (e) detecting
amplified DNA from Candida by hybridizing the amplified DNA with a
probe that selectively hybridizes with Candida DNA, the presence of
amplified DNA indicating systemic candidiasis.
In the method, the lysis step can use the lysis buffer from the
ISOQUICK.RTM. kit in addition to ZYMOLASE.RTM.-100T. The agitation
step can be by rocking at about 16 cycles per minute. The
extracting step can use the extraction matrix in the ISOQUICK.RTM.
kit. In the amplification step of the above method, one of the
primers of the primer pair is derived from the internal transcribed
spacer 1 (ITS1) and the other primer of the primer pair is derived
from the internal transcribed spacer 2 (ITS2). Alternatively, one
of the primers of the primer pair is derived from the internal
transcribed spacer 3 (ITS3) and the other primer of the primer pair
is derived from the internal transcribed spacer 4 (ITS4). The
detecting step hybridization can be by dot blot hybridization using
a genus or species specific Candida probe.
In the method of detecting systemic candidiasis, the DNA that is
amplified can be from C. albicans and the probe can specifically
hybridize with a specific nucleotide sequence of the nucleic acid
of SEQ ID NO: 5 as described in Example 2. By using the other
specific nucleic acids as provided herein, the method of Example 2
can be used to detect any of the other Candida species as taught
herein. If the DNA that is amplified is from C. parapsilosis, the
probe specifically hybridizes with a specific nucleotide sequence
of the nucleic acid of SEQ ID NO: 6. If the DNA that is amplified
is from C. tropicalis, the probe specifically hybridizes with a
specific nucleotide sequence of the nucleic acid of SEQ ID NO: 7.
If the DNA that is amplified is from C. glabrata, the probe
specifically hybridizes with a specific nucleotide sequence of the
nucleic acid of SEQ ID NO: 8. If the DNA that is amplified is from
C. krusei, the probe specifically hybridizes with a specific
nucleotide sequence of the nucleic acid of SEQ ID NO: 9. A nucleic
acid having homology with more than one Candida species can also be
used as a probe that specifically hybridizes with Candida DNA to
detect systemic candidiasis.
Additionally, it is contemplated that the nucleic acids (e.g.,
probes and primers) can be attached to or labeled with (covalently
or non-covalently) a detectable moiety. The probes may be suitably
labeled using, for example, a radio label, enzyme label,
fluorescent label, biotin-avidin label and the like for subsequent
visualization in the example of the dot blot hybridization
procedure taught in Example 2. An example of such a labeled nucleic
acid is the digoxigenin-UTP labelled probe provided in Example 2,
although others can be readily generated using standard methods
(See, e.g., Sambrook et al., 1989). The nucleic acids specific for
a given Candida species can each be labeled with a distinct
detectable moiety, such that species specific probes for several
species can be used with the same sample of amplified DNA to permit
species specific diagnosis. The distinct label for each species
specific probe can be detected in the sample if DNA from the
particular species is present in the subject.
The detection of fungal DNA as described herein can also be
performed using a ligase chain reaction (LCR). Essentially, this
reaction, known to those of skill in the art, involves the use of,
for each region to be detected, two primers that hybridize to the
same strand of the target DNA, either abutting each other or with
one or two nucleotides between the two primer sequences (i.e.,
"immediately 5'" or "immediately 3'" to the junction). The ligase
reaction is performed, and the products are electrophoresed through
a gel that can detect very small fragments, such as an
SDS-polyacrylamide gel. A positive result is one in which a product
equal in size to the sum of the two primers is produced, as this
indicates the presence of all of the target DNA region. It is
preferable that three reactions be run in three separate tubes,
targeted at detecting (1) the first Junction, (2) the second
junction and (3) an internal sequence as a positive LCR control. If
one wants to electrophorese all LCR products together through the
gel, primers must be carefully chosen such that their individual
sizes can be distinguished from the predicted size of any LCR
products. Alternatively, the product of each reaction can be
electrophoresed separately. Primers are preferably exactly
homologous to the target region and of a size between approximately
20-40 nucleotides.
The following examples are intended to illustrate, but not limit,
the invention. While they are typical of those that might be used,
other procedures known to those skilled in the art may be
alternatively employed.
EXAMPLES
Example 1
Nucleotide Sequence Analysis of the ITS2 Region of Candida albicans
and Related Species
Yeast strains and maintenance
All Candida isolates have been previously characterized by
assimilation (API) profiles and morphology (Van der Walt and
Yarrow, 1984). In addition, all C. albicans and C. parapsilosis
isolates have previously been electrophoretically karyotyped and
are known to represent distinct, non-related strains (Lasker et
al., 1989). All isolates were grown and maintained on
yeast-peptone-dextrose (YPD) medium (Guthrie and Fink, 1991). For
DNA extractions, 10 ml of overnight cultures grown on YPD at
37.degree. C. were washed twice in 1.times.TE buffer and the DNA
extracted by standard procedures (Sambrook et al., 1989). Prior to
PCR amplifications, DNA was digested with EcoRI restriction
endonuclease (New England Biolabs), electrophoresed on 1.0% agarose
gels, and stained with ethidium bromide (EtBr) to verify
concentration and purity.
PCR amplification and DNA sequencing
Taq polymerase, buffers, and conditions for PCR were those supplied
by the vendor (Perkin-Elmer/Cetus), using 100 ng genomic DNA per
reaction. For primary amplifications, 35 cycles of 95.degree. C.,
55.degree. C., and 72.degree. C. at one min. intervals were
followed by a five min. final extension at 72.degree. C. The
following "universal" ITS primers were used, for which calculated
Tm's have previously been reported (White et al., 1990):
ITS1 5' TCC GTA GGT GAA CCT GCG G 3' (SEQ ID NO: 1)
ITS3 5' GCA TCG ATG AAG AAC GCA GC 3' (SEQ ID NO: 2)
ITS4 5' TCC TCC GCT TAT TGA TAT GC 3' (SEQ ID NO: 3)
Primer ITS1 is to a conserved 3' domain in the 18S nuclear subunit.
Primer ITS3 is approximately 25 bp from the end of the 5.8S
subunit, and ITS4 is a reverse primer to a conserved region of the
nuclear large rDNA. In addition, a -21M13 forward primer sequence
(Messing et al. 1981) was added at the 5' end to primers ITS1 and
ITS4 for sequencing in the forward and reverse directions,
respectively, and consisted of the sequence: 5' GTA AAA CGA CGG CCA
G 3' (SEQ ID NO: 10) where the terminal 5' T of ITS1 and ITS4 made
17 bp of the 18 bp annealing sequence. From preliminary experiments
it was determined that the addition of this sequence did not alter
the nature of the derived PCR product. The aqueous phase of the
primary PCR reaction was ethanol-precipitated, dried, and
resuspended in 8 .mu.l TE buffer. The entire amount was loaded into
single wells of a 1.5% agarose, 1.0% NuSieve agarose gel (Lehmann
et al. 1992), electrophoresed at 110 V., and stained with EtBr.
Single, intensely staining bands of the appropriate size were
excised and the DNA was extracted in Spin-X cellulose acetate
columns (Costar, Inc.) for 30 min. at 40.degree. C., 13000.times.g.
The DNA was then ethanol-precipitated, washed twice in 70% ET/OH,
dried briefly, and resuspended in H.sub.2 O for sequencing.
Automated DNA sequencing (Smith et al. 1986), was performed using
the Applied Biosystems Catalyst 800 workstation, with the "Prism"
dye-primer dideoxy-sequencing reactions (Sanger et al. 1977), using
conditions supplied by the vendor (Applied Biosystems). The
precipitated DNA was dried and resuspended in 6 .mu.l of
formamide/50 mM EDTA (5:1), denatured for 2 min. at 90.degree. C.
and loaded on an Applied Biosystems model 373A DNA sequencer. All
DNAs were sequenced in both forward and reverse orientations, and
multiple runs were performed for all species and most strains
within a given species.
5.8s rDNA
5.8S sequence alignments were performed both manually and with the
"pileup" program from the University of Wisconsin Genetics Computer
Group (GCG) package (Devereux et al., 1984). ITS alignments were
performed in all possible pairwise combinations using the Needleman
and Wunsch algorithm as implemented by GCG (Needleman and Wunsch
1970). DNA parsimony and bootstrap analysis was performed using the
"Phylip" programs of Felsenstein (Felsenstein 1982), implemented on
a micro-vax (Digital Equip. Corp.) cluster. Dendrograms were
constructed using the global option and using a variety of
different species as the outgroup (Felsenstein 1985). Other 5.8S
sequences were: Neurospora crassa, Schizosaccharomyces pombe,
Saccharomyces cerevisiae, Pneumocystis carinii, Fusarium sambucium,
Epichloe typhina, Cephalosporium acremonium, Lentinula edodes.
For C. albicans and C. parapsilosis, where multiple strains were
analyzed, there was complete nucleotide conservation within the
entire 159 bp 5.8S region. The greatest degree of diversity for the
species used in this study was found in the two relatively
unconserved regions between bp 79-85 and bp 118-136. The overall
average degree of diversity between the Candida species was
approximately three percent. The minimum degree of diversity was
found between C. tropicalis and C. parapsilosis, with a single C-A
transversion at bp 62. Interestingly, both C. albicans and C.
krusei contained A-G transitions in the termination consensus
TCATTT.
A phylogenetic analysis was performed with all known fungal 5.8S
sequences using strict parsimony as implemented by Felsenstein and
statistical bootstrap analysis (Felsenstein 1982; 1985). P. carinii
was used as the outgroup considering previous findings based on 18S
analysis using a larger database of eukaryotic organisms (Edman et
al. 1988). There were a total of 47 informative sites for the
number of fungal sequences compiled, including 4 single base pair
gaps. Re-analysis of the data set without gaps did not
significantly alter the tree topology. The cumulative number of
positive selections out of 100 total iterations is given for each
branch point. The derived tree does not differ significantly from
previous research using a weighted difference algorithm for 18S
sequences, and supports the view that these species are related
such that C. albicans, C. parapsilosis and C. tropicalis are more
closely aligned than C. krusei within a clade. Likewise, C.
glabrata appears more distantly related and can equally be placed
at a number of positions within the larger branch of yeast-like
fungi. It is generally accepted that values of 70 or greater out of
100 randomly tested samples will represent similar trees to a
significant degree of probability.
ITS2 rDNA
The sequences of the ITS2 regions for C. albicans, C. parapsilosis,
C. tropicalis, C. glabrata and C. krusei are shown in the Sequence
Listing as SEQ ID NOs: 5-9.
A total of ten C. albicans isolates, representing typical and
morphologically (or physiologically) atypical strains, were found
to be identical at the nucleotide level within the ITS region.
Similarly, five strains of C. parapsilosis, displaying a wide range
of electrophoretic karyotypes and randomly amplified polymorphisms
(RAPD), were also identical to the type strain for the species. The
entire length of the ITS region was found to be species
specific.
Similar to the results of the 5.8S alignments, we found that C.
albicans, C. parapsilosis, and C. tropicalis were also most
homologous in this ITS region. This homology extended for the first
57 bp 5' immediately adjacent to the termination of the 5.8S
sequence. In contrast, the 3' region displayed little homology. For
C. krusei and C. glabrata there was no apparent homology either to
each other or to members of the C. albicans group over this entire
ITS region. Sequences were aligned in all possible pairwise
combinations (Needleman and Wunsch 1970), and the average degree of
similarity was found to be approximately 40 percent.
Analysis of the ITS2 region has revealed that C. albicans, and
possibly other closely related species, displays no interstrain
variation. In this respect this species resembles the opportunistic
fungus Cryptococcus neoformans, and is unlike the plant pathogen
Fusarium sambucinum which displays variation in this region.
Example 2
Detection of DNA from Candida albicans Cells in Blood by Use of the
Polymerase Chain Reaction (PCR)
Growth of C. albicans
C. albicans strain 36B was grown on Sabouraud's dextrose agar
Emmons slants for 48 h at 25.degree. C. Cells were harvested by
washing each slant with 5 ml of 0.85% NaCl, centrifuged at
1500.times.g for 10 min, and resuspended to the appropriate
concentration in freshly collected rabbit's blood or 0.85%
saline.
Yeast cell lysis and DNA purification
Blood from adult female rabbits (New Zealand White, Myrtle's Rabbit
Farm) was collected from the central ear artery into ISOLATOR
10.RTM. microbial tubes (Wampole Laboratories, Cranbury, N.J.)
containing an aqueous solution of 1 unit of purified saponin, 8
ml/L polypropylene glycol, 9,6 g/L Na polyanetholesulfonate and 16
g/L (Na).sub.2 EDTA; EDTA-coated tubes (Becton Dickinson,
Rutherford, N.J.); or heparinized tubes (Becton Dickinson). C.
albicans strain 36B (Quebec Gynecological Institute, Montreal,
Quebec) cells were then introduced and samples were centrifuged at
3000.times.g for 30 min. Supernatants were removed and an equal
volume of deionized water was added to lyse residual blood cells.
Remaining C. albicans cells were washed in 0.85% NaCl and pelleted
by centrifugation at 1500.times.g for 10 min. ISOLATOR 10.RTM.
tubes have proven superior to other blood collection systems for
the recovery of viable C. albicans cells from blood (Jones, 1990).
The use of the ISOLATOR 10.RTM. tubes for blood collection resulted
in PCR amplification of candidal DNA whereas the use of EDTA- or
heparin-coated tubes did not.
C. albicans DNA was extracted and purified using the ISOQUICK.RTM.
nucleic acid extraction kit according to the manufacturer's
instructions with the addition of ZYMOLASE.RTM.-100T, to allow its
use with fungi, since the ISOQUICK.RTM. kit was developed by
MicroProbe Corporation for the isolation and purification of DNA
from only mammalian cells and gram negative bacteria. Briefly,
pelleted cells were suspended in 100 .mu.l of sample buffer for 15
min after which 100 .mu.l of lysis buffer was added. The mixture
was incubated at 25.degree. C. for 1 h. Selected samples contained
ZYMOLYASE (ZYMOLYASE.RTM.-100T, Seikagaku Corp., Tokyo, Japan; 5
mg/ml in 1.0M sorbitol, 0.1M trisodium citrate, and 0.1%
2-mercaptoethanol) during the lysis step and were rocked at 16
cycles per min to optimize breakage of C. albicans cells. The
addition of ZYMOLYASE.RTM.-100T to the lysis step allowed for
successful adaptation of the ISOQUICK.RTM. kit for use with C.
albicans cells. Alternatively, C. albicans cells were disrupted
using a mini bead beater (Biospec Products, Bartlesville, Okla.)
(Glee et al. 1987). Cells (1 ml) were delivered into Sarstedt
microfuge tubes containing 1 ml of 0.5 mm diameter glass beads and
beaten at maximum speed for 2 min. A third method released C.
albicans DNA by boiling 1.times.10.sup.7 cells per ml in 2 mls of
deionized water in an Eppendorf microcentrifuge tube for 30 min.
Mechanical disruption of C. albicans cells by bead beating or
boiling was less effective in producing PCR amplifiable DNA; these
methods may be too harsh, resulting in shearing or fragmentation of
DNA. For precipitation of the DNA sodium acetate and other
components of the ISOQUICK.RTM. kit were used as directed.
After lysis, DNA was purified with the extraction matrix provided
in the ISOQUICK.RTM. kit, precipitated with sodium acetate in the
presence of isopropanol, and the precipitated DNA was dried by
vacuum centrifugation for 15 min.
PCR amplification of genomic DNA
Universal fungal primer pairs, ITS1 and 2 or ITS3 and 4,
synthesized by the CDC core facility, and the GeneAmpR DNA
amplification reagent kit using native Taq DNA polymerase (250 U,
Perkin Elmer Cetus, Alameda, Calif.) were used for PCR
amplification of genomic DNA (Saiki et al. 1988). These primers
amplify DNA from all fungi and some parasites. Examples of the
ITS1, ITS2, ITS3 and ITS4 primers are shown in the Sequence Listing
as SEQ ID NOs: 1, 4, 2 and 3, respectively. The reaction consisted
of the following: 53.5 .mu.l of double distilled, sterile water, 10
.mu.l of 10X reaction buffer, 16 .mu.l of a mixture of equimolar
(1.25 mM) quantities of dATP, dCTP, dGTP, and dTTP, 5 .mu.l of 20
.mu.M ITS1 or 3, 5 .mu.l of 20 .mu.M ITS2 or 4, 10 .mu.l of target
DNA, 0.5 .mu.l of Taq polymerase, and 6 .mu.l of 25 mM MgCl.sub.2.
Samples were overlaid with mineral oil prior to placement in the
thermal cycler (Perkin Elmer Cetus) to minimize evaporation during
DNA amplification. Samples were initially denatured in the thermal
cycler at 95.degree. C. for 5 min. This was followed by 30 cycles
of: denaturation at 95.degree. C. for 1 min, annealing at
50.degree. C. for 2 min, and extension at 72.degree. C. for 1.5
min. Final extension occurred at 72.degree. C. for 5 min.
After amplification, mineral oil was discarded. An equal volume of
chloroform was added to the samples which were then centrifuged for
5 min at 4100.times.g to extract residual mineral oil. The aqueous
layer was removed and the DNA precipitated from it by adding 2
volumes of ice-cold 100% ethanol followed by incubation for 30 min
at -70.degree.C. Samples were then centrifuged for 1 min at
4100.times.g, the ethanol removed, the samples dried under vacuum,
and resuspended in 20 .mu.l of TE buffer (20mM Tris plus 1 mM EDTA,
pH 8.0). Amplified DNA was visualized after agarose (1% agarose
plus 1% Nu-Sieve in TE buffer) gel electrophoresis by ethidium
bromide staining or by dot blot hybridization analysis.
Dot blot hybridization
C. albicans strain 3307 DNA was used as a probe for the dot blot.
To make the probe, 20 ng of C. albicans 3307 genomic DNA was
PCR-amplified using ITS1 and ITS2 or ITS3 and ITS4 as primer pairs.
The PCR product was then electrophoresed on an agarose gel and the
resultant DNA band cut out of the gel. The product was extracted
from the gel by the freeze-squeeze method of Thuring et al (Thuring
et al., 1975). The DNA probe was labeled by incubating overnight
with digoxigenin-dUTP from a nonradioactive-DNA labeling and
detection kit according to the manufacturers instructions ("Genius"
kit, Boehringer Mannheim, Indianapolis, Ind.). Other genus or
species specific probes derived from the nucleic acids of SEQ ID
NOs: 5-9 can also be used in this method.
Samples were prepared for the dot blot (Kafatos et al., 1979,
Lasker et al., 1992) by diluting 10 .mu.l of C. albicans DNA to 25
.mu.l with TE buffer, adding NaOH to a final concentration of 0.3M,
and incubating for 10 min at 25.degree. C. An equal volume of 2.0M
ammonium acetate was then added to each sample on ice. Each sample
was then dotted under vacuum onto a nitrocellulose filter using a
dot blot apparatus (BioRad, Richmond, Calif.) according to the
manufacturer's instructions. The filter was then removed from the
apparatus and dried at 80.degree. C. under vacuum for 2 h. The
dried filter was placed in a plastic bag, sealed, and prehybridized
with single-stranded salmon sperm DNA (10 .mu.g/ml) overnight in a
65.degree. C. water bath.
The digoxigenin-labeled probe was denatured by boiling for 5 min,
added to the filter in the plastic bag, and placed in a 65.degree.
C. water bath overnight. The filter was then washed twice for 30
min each in citrated saline (0.3M NaCl with 0.03M sodium citrate,
pH 7.0) and 0.1% SDS at 60.degree. C. (Lasker et al., 1992). Washed
filters were incubated for 30 min at 25.degree. C. with an
anti-digoxigenin antibody (1:5000) labeled with alkaline
phosphatase. Chromogen (nitroblue tetrazolium salt and
5-bromo-4-chloro-3-indolyl phosphate) was added (Lasker et al.,
1992) and color developed for 6 h at 25.degree. C. in the dark.
"Booster" PCR amplification
"Booster" PCR amplification was performed by the method of Ruano et
al. (Ruano et al., 1989). Briefly, the same protocol as outlined
above was used, but after 15 cycles of PCR amplification, samples
were removed from the thermal cycler and fresh primers were added
to a final concentration of 40 .mu.M. The samples were then
returned to the thermal cycler for 15 additional cycles and final
extension. The level of sensitivity of detection of the PCR product
from cells introduced into blood was improved from 10.sup.5 cells
per ml to 10.sup.3 cells per ml as detected by ethidium bromide
stained agarose gels. However, the specificity of this system was
poor since the negative control became positive.
Detection of PCR amplified products from C. albicans in saline by
agarose gel electrophoresis
A comparison of C. albicans DNA isolated and purified from saline
using the ISOQUICK.RTM. kit alone to that obtained by the use of
ZYMOLYASE.RTM.-100T plus the kit was performed. C. albicans cells
(10.sup.7/ ml saline) were lysed at either 37.degree. C. or
25.degree. C. The combined use of ZYMOLYASE.RTM.-100T and the
ISOQUICK.RTM. kit (at either 25.degree. C. or 37.degree. C.)
resulted in enhanced recovery of purified DNA relative to the kit
alone.
To determine the sensitivity of the ZYMOLASE.RTM.-100 T plus
ISOQUICK.RTM. method for cell breakage and DNA purification, C.
albicans cells were then serially diluted in saline (10.sup.7 to
10.sup.1 cells per ml) before breakage. Ethidium bromide stained
agarose gels demonstrated that 10.sup.3 cells per ml could be
detected by this method. Based on these results, all subsequent
experiments used ZYMOLYASE.RTM.-100T followed by DNA purification
with the ISOQUICK.RTM. kit at 25.degree. C.
Detection of PCR amplified products of C. albicans in blood by
agarose gel electrophoresis
To determine if the ZYMOLYASE.RTM.-100T plus ISOQUICK.RTM. kit
method could be used to detect C. albicans in blood, 10.sup.7 C.
albicans cells per ml was introduced into freshly collected
rabbit's blood as described above. Blood was collected into one of
the following: ISOLATOR 10.RTM. microbial tubes, EDTA-coated tubes,
or heparinized tubes. Amplified DNA was detected in the samples
prepared from cells introduced into blood drawn into ISOLATOR
10.RTM. tubes only. No DNA was detected in samples where blood had
been drawn into either only EDTA- or only heparin-coated tubes.
The sensitivity of detection for C. albicans DNA in blood using the
ZYMOLYASE.RTM.-100T plus ISOQUICK.RTM. kit method was determined by
serially diluting C. albicans cells (10.sup.7 to 10.sup.1 cells per
ml) in blood drawn into ISOLATOR 10.RTM. tubes. Using agarose gel
electrophoresis and ethidium bromide staining, 10.sup.5 cell per ml
could be detected.
Dot blot hybridization for detection of PCR amplified products of
C. albicans in blood or saline.
In an effort to improve the sensitivity for detection of C.
albicans DNA, a comparison was performed of the ethidium
bromide-stained agarose gel method to a dot blot hybridization
method for the detection of the PCR product. The dot blot method
allowed detection of 10.sup.1 cells per ml in saline versus
10.sup.3 cells per ml detected by agarose gel electrophoresis and
ethidium bromide staining. The sensitivity for detection of the PCR
product of C. albicans cells introduced into blood was 10.sup.1
cells per ml by the dot blot method versus 10.sup.5 cells per ml
for ethidium bromide stained agarose gels detection. The probe used
for the above dot blot was C. albicans-specific. C. tropicalis DNA
and human placental DNA did not react in the dot blot, supporting
the specificity of the probe. Thus, the methods taught herein are
capable of detecting Candida DNA in clinical samples such as
blood.
Universal fungal primers as described herein provide the potential
for amplification of DNA from all fungi. However, by using a C.
albicans-specific DNA probe, as in the above-described dot blot
hybridization step, the test was specific for C. albicans. The dot
blot assay can be conducted using specific probes for other Candida
species, as described herein, or other fungi. Furthermore, because
the present method can gently extract DNA from clinical samples,
the method can also use vital, bacterial or other fungal primers
for the PCR reaction followed by specific DNA probes for each genus
or species in the dot blot as described above.
Throughout this application various publications are referenced
within parentheses. Full citations for these publications may be
found at the end of the specification immediately preceding the
Sequence Listing. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
REFERENCES
Armstrong, C. 1989. Problems in Management of Opportunistic Fungal
Diseases. Rev. Infect. Dis. 2: S1591-S1599.
Barns, S. M., Lane, D. J., Sogin M. L., Bibeau, C. and Weisburg, W.
G. (1991) Evolutionary relationships among pathogenic Candida
species and relatives. J. Bacteriol. 173: 2250-2255.
Buchman, T. G., M. Rosser, W. G. Merz, and P. Charache. 1990.
Detection of surgical pathogens by in vitro DNA amplification. Part
I. Rapid identification of Candida albicans by in vitro
amplification of a fungus-specific gene. Surgery 108: 338-347.
Dams, E., Hendriks, L., Van de Peer, Y., Neefs, J. and Smits, G.
(1988) Compilation of small ribosomal subunit RNA sequences. Nucl.
Acids Res. 16: r87-r174.
Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive
set of sequence analysis programs for the VAX. Nucl. Acids Res. 12:
387-397.
Edman, J. C., Kovacs, J. A., Masur, H., Santi, D. V., Elwood, H. J.
and Sogin, M. L. (1988) Ribosomal RNA shows Pneumocystis carinii to
be a member of the fungi. Nature (London). 334: 519-522.
Felsenstein, J. (1982) Numerical methods for inferring evolutionary
trees. Quart. Rev. Biol. 57: 379-404.
Felsenstein, J. (1985) Confidence limits on phylogenies: an
approach using the bootstrap. Evolution. 39: 783-791.
Glee, P. M., P. J. Russell, J. A. Welsch, J. C. Pratt, and J. E.
Cutler. 1987. Methods of DNA extraction from Candida albicans.
Anal. Biochem. 164: 207-213.
Guthrie, C. and Fink, G. R. (1991) Guide to yeast genetics and
molecular biology. Meth. Enzymol. 194: 3-20.
Jones, J. M. 1990. Laboratory diagnosis of invasive candidiasis.
Clin. Microbiol. Rev. 3: 32-45.
Kafatos, F. C., C. W. Jones, and A. Efstraliadis. 1979.
Determination of nucleic acid sequence homologies and relative
concentrations by a dot blot hybridization procedure. Nucl. Acids
Res. 3: 1541-1552.
Lasker, B. A., J. M. Brown, and M. M. McNeil. 1992. Identification
and epidemiological typing of clinical and environmental isolates
of the genus Rhodococcus with use of a digoxigenin-labeled rDNA
gene probe. Clin. Infect. Dis. 15: 223-233.
Lasker, B. A., Carle, G. F., Kobayashi, G. S. and Medoff, G. (1989)
Comparison of the separation of Candida albians chromosome-sized
DNA by pulsed-field gel electrophoresis techniques. Nucl. Acids
Res, 17: 3783-3793.
Lehmann, P. F., Lin, D. and Lasker, B. A. (1992) Genotypic
identification and characterization of species and strains within
the genus Candida by using random amplified polymorphic DNA. J.
Clin. Micro, 30: 3249-3254.
Magee, B. B., D'Souza, T. M. and Magee, P. T. (1987) Strain and
species identification by restriction fragment length polymorphisms
in the ribosomal DNA repeat of Candida species. J. Bacteriol. 169:
1639-1643.
Messing, J., Crea, R. and Seeburg, P. H. (1981) A system for
shotgun DNA sequencing. Nucl. Acids Res. 9: 309-319.
Mitchell, T. G., White, T. J. and Taylor, J. W. (1992) Comparison
of 5.8S ribosomal DNA sequences among the basidiomycetous yeast
genera Cystofilobasidium, Filobasidium and Filobasdiella. J. Med.
Vet. Mycol. 30: 207-218.
Miyakawa, y., T. Mabuchi, K. Kagaya, and Y. Fukagawa. 1992.
Isolation and detection of Candida albicans by polymerase chain
reaction. J. Clin. Micro. 30: 894-900.
Needleman, S. B. and Wunsch, C. D. (1970) A general method
applicable to the search for similarities in the amino acid
sequence of two proteins. J. Mol. Biol. 48: 443-453.
Odds, F. C. 1988. Candida and Candidosis: A Review and
Bibliography, 2nd Ed., Philadelphia: Bailere Tindall.
O'Donnell, K. (1992) Ribosomal DNA internal transcribed spacers are
highly divergent in the phytopathogenic ascomycete Fusarium
sambucinum (Gibberella pulicaris). Curr. Genet, 22: 213-220.
Ruano, G. W., W. Tenton, and K. K. Kidd. 1989. Biphasic
amplification of very dilute DNA samples via "booster" PCR. Nucl.
Acids Res. 3: 5407-5411.
Saiki, K. K., D. H. Gelfand, S. Stafford, S. J. Scharf, R. Higuchi,
G. F. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer directed
enzymatic amplification od DNA with thermostable DNA polymerase.
Science 239: 487-491.
Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A
laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring
Harbor, N.Y. 1989.
Sanger, F., Nicklen, S. and Coulson, A. R. (1977) DNA sequencing
with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74:
5463-5467.
Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C.,
Connell, C. R., Heiner, C., Kent, S. B. H. and Hood, L. E. (1986)
Fluorescence detection in automated DNA sequence analysis. Nature
(London). 321: 674-679.
Telenti, A., G. R. Roberts. 1989. Fungal blood cultures. Fur. J.
Clin. Microbiol. Infect. Dis. 8: 151-156.
Thrash-Bingham, C., and Gorman, J. A. (1992) DNA translocations
contribute to chromosome-length polymorphisms in Candida albicans.
Curt. Genet. 22: 93-100.
Thuring, R. W. J., J. P. Sanders, and P. Borst. 1975. A freeze
squeeze method for recovering long DNA from agarose gels. Anal.
Biochem. 66: 213-220.
Van der Walt, J. P. and Yarrow, D. (1984) Methods for the
Isolation, maintenance, classification and identification of
yeasts. in Kreger-van Rij, N. J. W. (Ed). The yeasts: A taxonomic
study. Elsevier, Amsterdam. pp. 45-104.
White, T. J., Bruns, T. D., Lee S. B. and Taylor, J. W. (1990)
Amplification and direct sequencing of fungal ribosomal RNA genes
for phylogenetics. in Innis, M. A., Gelfand, D. H., Sninsky, J. J.
and White, T. J. (Eds). PCR Protocols. A guide to methods and
applications. Academic Press, San Diego. pp. 315-322.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 10 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TCCGTAGGTGAACCTGCGG19 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GCATCGATGAAGAACGCAGC20 (2) INFORMATION FOR SEQ ID NO:3: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:3: TCCTCCGCTTATTGATATGC20 (2) INFORMATION FOR SEQ ID NO:4: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:4: GCTGCGTTCTTCATCGATGC20 (2) INFORMATION FOR SEQ ID NO:5: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 151 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:5:
CTCCCTCAAACCGCTGGGTTTGGTGTTGAGCAATACGACTTGGGTTTGCTTGAAAGACGG60
TAGTGGTAAGGCGGGATCGCTTTGACAATGGCTTAGGTCTAACCAAAAACATTGCTTGCG120
GCGGTAACGTCCACCACGTATATCTTCAAAC151 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 124 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:6:
CTCCCTCAAACCCTCGGGTTTGGTGTTGAGCGATACGCTGGGTTTGCTTGAAAGAAAGGC60
GGAGTATAAACTAATGGATAGGTTTTTTCCACTCATTGGTACAAACTCCAAAACTTCTTC120
CAAA124 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 141 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CTCCCTCAAACCCCCGGGTTTGGTGTTGAGCAATACGCTAGGTTTGTTTGAAAGAATTTA60
ACCGTGGAAACTTATTTTAAGCGACTTAGGTTTATCCAAAACGCTTATTTTGCTAGTGGC120
CACCACAATTTATTTCATAAC141 (2) INFORMATION FOR SEQ ID NO:8: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 231 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:8:
CCTTCTCAAACACATTGTGNTTGGTANTGAGTGATACNCNNTTTTGATNTAACTTNAAAT60
TGTAGGCCATATCAGTATGTGGGACACGAGNGCAAGCTTCTCTATTAATCTGCTGCTGCT120
TTGCGCGAGCGGCGGGGGTTAATACTCTATTAGGTTTTACCAACTCGGTGTTGATCTAGG180
GAGGGATAAGTGAGTGTTTTGTGCGTGCTGGGCAGACAGACGTCTTTAAGT231 (2)
INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 177 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:9:
GAGCGTCGTTTCCATCTTGCGCGTGCGCAGAGTTGGGTGAGCGGANGTACCGACGTGTAA60
AGAGCGTCGGAGCTGCGACTCNNCTGAAAGGGAGCNNANTGGCCCGAGCGAACTAGACTT120
TTTTTNAGGGNCCGTTTTGGGCCCCAGAACGNAGTTTTNCCNAGGNCAACAAAAAGN177 (2)
INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:10: GTAAAACGACGGCCAG16
__________________________________________________________________________
* * * * *